The present invention relates to a non-human animal model for obesity and uses of such an animal for studying and developing methods for modifying the peripheral as well as the central melanocortinergic pathways towards controlling body weight. In particular, the present invention relates to a proopiomelanocortin (Pomc) homozygous mutant mouse and uses thereof.
The regulation of body weight, and particularly, obesity and conditions related thereto, is a major health concern throughout the world, and particularly in the United States, contributing to morbidity and mortality. Obesity is a metabolic disorder characterized by excessive accumulation of fat stores in adipose tissue. In humans, its causes are a complex interplay of genetics, environment and culture. It is well known that a regimen of diet and exercise leading to weight loss is the best approach for treating obesity, but unfortunately, such regimens are frequently unsuccessful. Oftentimes, an individual""s inability to lose weight may be due to genetically inherited factors that contribute to increased appetite, a preference for high calorie foods, reduced physical activity and an abnormal metabolism. People inheriting or acquiring such predispositions are prone to obesity regardless of their efforts to combat the condition.
On the other side of the spectrum of body weight problems, other individuals suffer from one or more xe2x80x9cwastingxe2x80x9d disorders (e.g., wasting syndrome, cachexia, sarcopenia) which cause undesirable and/or unhealthy loss of weight or loss of body cell mass. In the elderly as well as in AIDS and cancer patients, wasting disease can result in undesired loss of body weight, including both the fat and the fat-free compartments. Wasting diseases can be the result of inadequate intake of food and/or metabolic changes related to illness and/or the aging process. Cancer patients and AIDS patients, as well as patients following extensive surgery or having chronic infections, immunologic diseases, hyperthyroidism, extraintestinal Crohn""s disease, psychogenic disease, chronic heart failure or other severe trauma, frequently suffer from wasting disease which is sometimes also referred to as cachexia, ametabolic and, sometimes, an eating disorder. Cachexia is additionally characterized by hypermetabolism and hypercatabolism. Although cachexia and wasting disease are frequently used interchangeably to refer to wasting conditions, there is at least one body of research which differentiates cachexia from wasting syndrome as a loss of fat-free mass, and particularly, body cell mass (Mayer, 1999, J. Nutr. 129(1S Suppl.): 256S-259S). Sarcopenia, yet another such disorder which can affect the aging individual, is typically characterized by loss of muscle mass. End stage wasting disease as described above can develop in individuals suffering from either cachexia or sarcopenia.
In addition to the obvious health risks associated with being overweight or underweight, the tangential detrimental effects of such conditions are equally troublesome. For the obese individual, health effects can include a myriad of physical conditions related to, or affected by, excess body weight (e.g., cardiovascular disease, diabetes, cancer, hypertension, etc.) as well as physiological damage due to an overweight person""s loss of self-esteem, depression, etc. For the underweight individual, conditions related to or affected by low body weight can include heart failure, susceptibility to infectious disease as a result of immune system weakness, and depression. Moreover, the rise in bulemia and anorexia in the past few decades is alarming, and illustrates the disturbing emphasis on ideal body size and shape regardless of the severe health consequences.
Radical treatments to treat obesity include surgical procedures such as liposuction and stomach stapling. In addition, numerous drugs have been utilized in an effort to regulate a person""s metabolism and/or to decrease appetite. Many of such drugs, however, have demonstrated harmful effects and have since been taken off of the market. Other replacement drug therapies have proven less effective, and the long term health consequences of such drugs are unknown. For the underweight individual, who may be suffering from undesired weight loss due to a disease such as cancer or AIDS, efforts to maintain or gain weight can be equally problematic.
Faced with such a long felt, but unsolved need for simple and effective methods for regulating body weight, researchers, over the last several decades, have expended literally hundreds of millions of dollars to investigate compounds that can be used to treat body weight problems such as obesity without the negative implications experienced with other, previously tested, weight regulating drugs. While altering appetite can affect weight, so can the regulation of the fat stores in adipose tissue. This latter approach has been an under-appreciated field relative to regulation of appetite. For instance, compared to the list of compounds directed at inhibition of energy uptake (appetite suppressants), very few compounds have been identified which stimulate fat mobilization or suppress lipid sequestration.
Physiologists have postulated for years that, when a mammal overeats, the resulting excess fat stores signal to the brain that the body is obese which, in turn, causes the body to eat less and burn more dietary fat. G. R. Hervey, Nature (London), 227:629-631 (1969). This model of feedback inhibition is supported by parabiotic experiments, which implicates circulating hormones controlling adiposity. Genetic studies in model organisms, especially the mouse, have allowed the identification of molecules important for the regulation of body weight. These include leptin (Zhang et al., 1995, Nature 372:425-432, incorporated herein by reference in its entirety), a leptin receptor (Tartaglia et al., 1995, Cell 83:1263-1271) and a melanocortin receptor (Huszar et al., 1997, Cell 88:131-141).
Findings from several lines of investigations have placed proopiomelanocortin (Pomc) and the peptides derived from it at a pivotal position in the central pathways for energy homeostasis. Obesity in the autosomal dominant lethal yellow (Ay/a) mouse, for example, is caused by ectopic expression of the agouti protein in the brain, where it antagonizes the melanocortin receptor 4 (MC4-R), a receptor found within the central nervous system (Lu et al., 1994, Nature 371:799-802). Agouti-related protein (AgRP) is normally expressed in the brain and antagonizes MC4-R. In transgenic mice, overexpression of AgRP results in obesity (Graham et al, 1997, Nat. Genet. 17:273-274 and Ollmann et al., 1997, Science 278:135-138). Targeted deletion of the MC4-R produces obesity similar to that of Ay mice, which is characterized by adult onset obesity and increased linear growth (Huszar et al., 1997, Cell 88:131-141). Pharmacological evidence has further suggested the importance of a melanocortinergic pathway in the central regulation of energy balance: decreased feeding was observed after central administration of an MC4-R agonist (xcex1-MSH analog) to normal mice and increased feeding after central administration of a synthetic MC4-R antagonist to normal mice when measured for 12 hours (Fan et al., 1997, Nature 385:165-168).
Understanding of the regulation of fat stores was greatly advanced by the discovery of leptin, the gene affected in the obese (ob) mutation. Leptin is secreted by adipose tissue, and its levels increase with increasing fat stores. Leptin is known to have both central and peripheral effects. There are high affinity receptors for leptin in the hypothalamus. Absence of either leptin or the leptin receptor leads to morbid obesity, presumably because the hypothalamus receives no fat signal, and accordingly acts as if the animal is completely without fat stores, and in some manner directs adipocytes to accumulate fat. The use of leptin to treat obesity in mice, however, requires very high, non-physiological doses. Thus, leptin alone has not been found to be a particularly useful anti-obesity agent.
To treat wasting and cachexia in patients such as the elderly, AIDS patients and cancer patients, anabolic steroids, growth hormone, dietary regimens, erythropoietin, cytokine therapy and anti-cytokine therapy, among other therapies, have been used to try to improve the condition of such patients. Such therapies cross a wide range of target cells, may have undesirable systemic side effects, may require toxic doses to work, and may not be sufficient to completely address the complex biological dysfunction related to different types of wasting disorders, however, and therefore, research is ongoing in the effort to find additional solutions to this problem.
Therefore, there remains a need in the art for a simple, safe and effective method for controlling body weight and for treating conditions related to or caused by undesired, health-compromising body weight.
The development of transgenic and xe2x80x9cknock-outxe2x80x9d animal technology has provided significant advances for obtaining more complete information about complex systems in vivo. By manipulating the expression of gene(s) in vivo, it is possible to gain insight into the roles of such genes in a particular system or to study aspects of the system in a genetically controlled environment. The biochemical activities associated with obesity in a small mammal such as the mouse, will allow analysis of the disorder, and conditions related thereto, at molecular and cellular levels that are often impossible to analyze in humans.
The present invention generally relates to a non-human animal model for studying and developing protocols for modifying the peripheral as well as the central melanocortinergic pathways controlling body weight. In particular, the present invention relates to a genetically modified non-human animal comprising a genetic modification within at least one allele of its Pomc locus, wherein the genetic modification results in a reduction in proopiomelanocortin (Pomc) peptide action in the animal. In one embodiment, the present invention relates to a POMC homozygous mutant mouse model. Such animal models are used for studying and developing protocols for modifying the peripheral as well as the central melanocortinergic pathways controlling body weight in Pomc mutants, for studying and developing protocols for controlling other forms of weight dysregulation, and for determining which of the specific POMC compounds are most efficient at mediating the effects on weight loss or weight gain and on adrenal insufficiency.
More particularly, one embodiment of the present invention relates to a genetically modified non-human animal useful for studying peripheral and central pathways of energy homeostasis. The genetically modified non-human animal comprises a genetic modification within at least one allele of its Pomc locus, wherein the genetic modification results in a reduction in proopiomelanocortin (Pomc) peptide action in the animal (e.g., a heterozygous mutant animal). In one embodiment, the genetic modification includes, but is not limited to, a deletion, an insertion, a substitution and/or an inversion of nucleotides in the Pomc locus. The genetic modification can be a modification including or within exon 3 of the Pomc locus which results in a reduction in Pomc peptide action, or a modification in a region of the Pomc locus other than exon 3 which results in a reduction in Pomc peptide action (e.g., exon 1, exon 2 and/or a regulatory region of the Pomc locus). In a preferred embodiment, the genetic modification is a deletion of a nucleic acid sequence within at least one allele of the Pomc locus, wherein the deletion results in an reduction of expression of Pomc peptides by the animal. In another embodiment, the animal comprises a genetic modification within two alleles (i.e., both alleles) of the Pomc locus, wherein the genetic modification results in an absence of Pomc peptide action in the animal (e.g., a homozygous mutant animal). Preferably, the genetic modification is a deletion of a nucleic acid sequence within both alleles of the Pomc locus, wherein the deletion results in an absence of expression of Pomc peptides by the animal.
In one embodiment of the present invention, the genetic modification is a deletion of a nucleic acid sequence comprising exon 3 of Pomc. In another embodiment, the genetic modification is a deletion of exon 3 of Pomc. In yet another embodiment, the genetic modification is a deletion of a portion of exon 3 of Pomc sufficient to reduce or prevent expression of Pomc peptides by at least one allele and more preferably, by both alleles, of the Pomc locus of the animal. In further embodiments of the present invention, the genetically modified non-human animal is characterized by a phenotypic characteristic which includes, but is not limited to obesity, a defect in adrenal development, altered pigmentation, measurably increased serum leptin levels, increased food uptake, and/or measurably reduced serum levels of a hormone selected from the group of corticosterone, aldosterone and epinephrine, as compared to a wild-type sibling of the animal.
In one embodiment of the present invention, the genetically modified non-human animal is a mouse. In this embodiment, the genetic modification is preferably a deletion from the genome of a nucleic acid sequence comprising SEQ ID NO:7, although other modifications as discussed above, which result in a reduction in the action of Pomc peptides, are encompassed by the present invention.
Another embodiment of the present invention relates to a method to study the molecular and biochemical events associated with body weight gain and loss, and particularly, with such events that are associated with obesity. More particularly, such a method includes the steps of: (a) harvesting cells, tissues or body fluids from a genetically modified non-human animal of the present invention; and, (b) comparing the cells, tissues or body fluids from the genetically modified non-human animal to cells, tissues or body fluids from a wild-type sibling of the genetically modified non-human animal. In one embodiment, the step of comparing is performed by an assay selected from the group consisting of morphological examination of the cells, tissues or body fluids; histological examination of the cells, tissues or body fluids; evaluation of Pomc peptide biological activity in the animal; evaluation of free fatty acid metabolism in the animal; evaluation of lipolysis and fatty acid sequestration in the animal; evaluation of weight gain or loss in the animal; evaluation of hormone levels in the animal; and, evaluation of blood biochemistry in the animal.
Yet another embodiment of the present invention relates to a method to identify POMC compounds, and particularly, homologues and mimetics, for use in regulating peripheral and central pathways of energy homeostasis. The method includes the steps of: (a) administering a compound to be evaluated to a genetically modified non-human animal which comprises a genetic modification within two (both) alleles of its Pomc locus, wherein the genetic modification results in an absence of Pomc peptide action in the animal; and, (b) evaluating physiological and pathological changes in the genetically modified non-human animal as compared to a non-human animal selected from the group of: (i) a second genetically modified non-human animal comprising a genetic modification within at least one allele of its pomc1 locus, wherein the genetic modification results in a reduction in proopiomelanocortin (Pomc) peptide action in the animal; and (ii) a third non-human animal having a genome comprising a wild-type pomcl locus at two (i.e., both) alleles. Various compounds which can be evaluated include, but are not limited to POMC compounds, including a Pomc peptide (i.e., a peptide encoded by the Pomc gene), a fragment of such a peptide (including both biologically active and inactive fragments), a homologue of such a peptide, a mimetic (peptide or non-peptide) of such a peptide, a fusion protein comprising such a peptide, and any pharmaceutical salts of such a peptide, as well as any non-Pomc peptide.
Yet another embodiment of the present invention relates to a method of producing a genetically modified non-human animal useful for studying peripheral and central pathways of energy homeostasis. Such a method includes the steps of: (a) introducing into an embryonic stem cell of a non-human animal a targeting vector comprising a Pomc locus containing a modification of a nucleic acid sequence sufficient to result in a reduction in proopiomelanocortin (Pomc) peptide action in the animal; and, (b) obtaining progeny having the modification stably incorporated into their genome, wherein the modification results in a reduction in expression of proopiomelanocortin (Pomc) peptides by the animal. In one embodiment, this method additionally includes the step of obtaining progeny having the modification stably incorporated into their genome, wherein the modification results in an absence of expression of Pomc peptides by the animal. In a preferred embodiment, the modification is a deletion in a nucleic acid sequence sufficient to result in the reduction of Pomc peptide action in the animal.
Another embodiment of the present invention is a genetically modified mouse useful for studying peripheral and central pathways of energy homeostasis. The mouse is produced by a method comprising the steps of: (a) isolating from a source of murine genomic DNA a nucleic acid molecule comprising SEQ ID NO:7; (b) modifying a nucleic acid sequence comprising SEQ ID NO:7 in the nucleic acid molecule to form a genetically modified nucleic acid molecule; (c) inserting a selectable marker into the genetically modified nucleic acid molecule to create a targeting vector; (d) transfecting the targeting vector into embryonic stem cells; (e) selecting embryonic stem cells from step (d) which have incorporated the targeting vector at a target locus comprising SEQ ID NO:7 by homologous recombination; (f) inserting the embryonic stem cells comprising the targeting vector into non-human animal blastocysts; and, (g) impregnating a female surrogate with the non-human animal blastocysts to produce the genetically modified mouse. In a preferred embodiment, the modification of step (b) is a deletion in the nucleic acid sequence comprising SEQ ID NO:7.